Bioconjugation Techniques and Experimental Processing of Myeloperoxidase Detection System

Cardiovascular disease (CVD) is the leading cause of death in the United States since the mid-20th century and has many well-established biomarkers such as C-reactive protein and N terminal pro-8 type peptide. Recent studies suggest that detection of a specific enzyme, myeloperoxidase (MPO) can be used for improved risk stratification in CVD, independent of other more established biomarkers. Myeloperoxidase is an enzyme produced by leukocytes, and functions as a catalyst for the creation of reactive oxidants and radical species. The pathways utilizing MPO have been determined to be an important process in phagocytosis. However, these same pathways are identified as potentially proatherogenic biological activities at various stages of CVD development.Measurement of MPO appears to be a valuable tool in the assessment of early stages of CVD, and this study investigated the viability of an electrochemical sensor system to detect MPO. Specifically, this system used a single-use electrochemical sensor prototype, with a bio-recognition mechanism using MPO antibody. The fabrication and preparation of the sensor system explored two separate bioconjugation techniques, 2 iminothiolane (Traut’s reagent) and N-succinimidyl S-acetylthioacetate (SATA), to immobilize the MPO antibody to the gold working electrode. Bioconjugation procedures for the immobilization of the MPO antibody were established, and the results of performance of these bioconjugated MPO antibody sensors were presented and discussed. Electrochemical impedance spectroscopy over a frequency range of 0.01 Hz – 10,000 Hz for a bioconjugated MPO antibody electrode was carried out to assess the surface coverage of the electrode element by the antibody. Preliminary measurements of MPO enzyme using this bioconjugated antibody-sensor appeared to be feasible, and a lower detection limit at a concentration of 0.008 µg/ml MPO was observed in this study.